Bottom Line:
We report that LprG expressed in Mtb binds to lipoglycans, such as lipoarabinomannan (LAM), that mediate Mtb immune evasion.Lipoglycan binding to LprG was dependent on both insertion of lipoglycan acyl chains into a hydrophobic pocket on LprG and a novel contribution of lipoglycan polysaccharide components outside of this pocket.An lprG mutant (Mtb ΔlprG) had lower levels of surface-exposed LAM, revealing a novel role for LprG in determining the distribution of components in the Mtb cell envelope.

ABSTRACTMycobacterium tuberculosis (Mtb) virulence is decreased by genetic deletion of the lipoprotein LprG, but the function of LprG remains unclear. We report that LprG expressed in Mtb binds to lipoglycans, such as lipoarabinomannan (LAM), that mediate Mtb immune evasion. Lipoglycan binding to LprG was dependent on both insertion of lipoglycan acyl chains into a hydrophobic pocket on LprG and a novel contribution of lipoglycan polysaccharide components outside of this pocket. An lprG mutant (Mtb ΔlprG) had lower levels of surface-exposed LAM, revealing a novel role for LprG in determining the distribution of components in the Mtb cell envelope. Furthermore, this mutant failed to inhibit phagosome-lysosome fusion, an immune evasion strategy mediated by LAM. We propose that LprG binding to LAM facilitates its transfer from the plasma membrane into the cell envelope, increasing surface-exposed LAM, enhancing cell envelope integrity, allowing inhibition of phagosome-lysosome fusion and enhancing Mtb survival in macrophages.

ppat-1004471-g003: LprG V91W hydrophobic pocket mutation reduces binding of Mtb glycolipids and lipoglycans.LprG-V91W was immobilized on a CM5 sensor chip, and sensograms were obtained as in Fig. 2 for (A) PIM2 (B) PIM6 (C) LM and (D) ManLAM. Results are from one experiment and representative of three independent experiments.

Mentions:
To assess the relative contributions of the hydrophobic binding pocket vs. other sites on LprG, we studied glycolipid and lipoglycan binding to LprG-V91W, which has a mutated pocket that precludes binding of the acyl chains of triacylated glycolipids [31]. PIM2 (Fig. 3A) and PI-LAM failed to bind to LprG-V91W, but PIM6, LM and ManLAM bound to LprG-V91W with an affinity approximately 10- to 130-fold lower than their affinity for LprG (Fig. 3B–D) (Table 1). The loss of acyl-chain dependent interaction with LprG-V91W increased substrate dissociation rates (Koff), indicating less stable interactions. These results indicate the presence of novel interactions of a subset of glycolipids/lipoglycans with LprG at a site outside of the hydrophobic pocket. Moreover, this suggests that these interactions include glycolipid and lipoglycan structures other than the acyl chains that bind within the hydrophobic pocket of LprG, consistent with the implication of novel contributions of saccharide structures of substrates in interactions with LprG.

ppat-1004471-g003: LprG V91W hydrophobic pocket mutation reduces binding of Mtb glycolipids and lipoglycans.LprG-V91W was immobilized on a CM5 sensor chip, and sensograms were obtained as in Fig. 2 for (A) PIM2 (B) PIM6 (C) LM and (D) ManLAM. Results are from one experiment and representative of three independent experiments.

Mentions:
To assess the relative contributions of the hydrophobic binding pocket vs. other sites on LprG, we studied glycolipid and lipoglycan binding to LprG-V91W, which has a mutated pocket that precludes binding of the acyl chains of triacylated glycolipids [31]. PIM2 (Fig. 3A) and PI-LAM failed to bind to LprG-V91W, but PIM6, LM and ManLAM bound to LprG-V91W with an affinity approximately 10- to 130-fold lower than their affinity for LprG (Fig. 3B–D) (Table 1). The loss of acyl-chain dependent interaction with LprG-V91W increased substrate dissociation rates (Koff), indicating less stable interactions. These results indicate the presence of novel interactions of a subset of glycolipids/lipoglycans with LprG at a site outside of the hydrophobic pocket. Moreover, this suggests that these interactions include glycolipid and lipoglycan structures other than the acyl chains that bind within the hydrophobic pocket of LprG, consistent with the implication of novel contributions of saccharide structures of substrates in interactions with LprG.

Bottom Line:
We report that LprG expressed in Mtb binds to lipoglycans, such as lipoarabinomannan (LAM), that mediate Mtb immune evasion.Lipoglycan binding to LprG was dependent on both insertion of lipoglycan acyl chains into a hydrophobic pocket on LprG and a novel contribution of lipoglycan polysaccharide components outside of this pocket.An lprG mutant (Mtb ΔlprG) had lower levels of surface-exposed LAM, revealing a novel role for LprG in determining the distribution of components in the Mtb cell envelope.

ABSTRACTMycobacterium tuberculosis (Mtb) virulence is decreased by genetic deletion of the lipoprotein LprG, but the function of LprG remains unclear. We report that LprG expressed in Mtb binds to lipoglycans, such as lipoarabinomannan (LAM), that mediate Mtb immune evasion. Lipoglycan binding to LprG was dependent on both insertion of lipoglycan acyl chains into a hydrophobic pocket on LprG and a novel contribution of lipoglycan polysaccharide components outside of this pocket. An lprG mutant (Mtb ΔlprG) had lower levels of surface-exposed LAM, revealing a novel role for LprG in determining the distribution of components in the Mtb cell envelope. Furthermore, this mutant failed to inhibit phagosome-lysosome fusion, an immune evasion strategy mediated by LAM. We propose that LprG binding to LAM facilitates its transfer from the plasma membrane into the cell envelope, increasing surface-exposed LAM, enhancing cell envelope integrity, allowing inhibition of phagosome-lysosome fusion and enhancing Mtb survival in macrophages.